In short: recombinant DNA technology joins DNA from different sources and multiplies it inside a host cell. The exam is less about reciting the steps than about the decisions — which vector, which restriction site, which screen — and about knowing what each screening result actually proves. FLN Tutors (Freelancer Network) also offers one-to-one help with the same topics. Matched to a verified specialist who knows your course, your university's marking criteria, and your deadline.
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Every set of notes lists the same five steps. Listing them earns the first mark or two; explaining the choice inside each one earns the rest.
| Step | What happens | The decision being tested |
|---|---|---|
| 1. Isolate the DNA | Extract the gene of interest and the vector | Genomic DNA or cDNA, and why |
| 2. Cut | Digest both with restriction enzymes | Which enzyme, and which site on the vector |
| 3. Join | Ligate insert into vector with DNA ligase | Sticky or blunt ends; how to stop self-ligation |
| 4. Transfer | Introduce the construct into a host | Which host, and which transfer method |
| 5. Select and screen | Find the cells that carry what you wanted | Which screen, and what it proves |
The single most common exam weakness is answering step 5 as though selection and screening were the same thing. They are not. Selection kills the cells you do not want, usually with an antibiotic. Screening lets you look at surviving cells and tell them apart. An antibiotic plate selects; blue-white screens.
This page concentrates on steps 2, 4 and 5, because that is where the reasoning is. For ligation ratios and restriction mapping see biotechnology help; for PCR, primer design and gel electrophoresis see molecular biology help.
A vector must be able to replicate independently in the host, must carry at least one selectable marker, must have unique restriction sites where an insert can go, and must be small enough to handle and take up efficiently.
| Vector | Rough insert capacity | Typically used for |
|---|---|---|
| Plasmid | Up to around 10 kb | Routine cloning of genes |
| Bacteriophage lambda | Roughly 10 to 20 kb | Genomic libraries |
| Cosmid | Roughly 35 to 45 kb | Larger genomic fragments |
| BAC | Over 100 kb | Genome projects |
| YAC | Several hundred kb | Very large fragments |
Quote capacities as approximate ranges, because the exact figures differ between textbooks and an examiner marks against the one you were taught.
Cloning vector or expression vector? This distinction is asked constantly and answered badly. A cloning vector exists to maintain and copy a piece of DNA — you want the sequence, not the protein. An expression vector additionally carries everything needed to transcribe and translate that insert in the host: a promoter the host recognises, a ribosome binding site, and usually a controllable induction system. Every expression vector is a cloning vector; the reverse is not true. If a question asks you to produce a protein and your answer stops at a cloning vector, the answer is incomplete.
This is the classic question, and it is reasoning rather than recall.
The vector. pBR322, about 4.4 kb, carrying two antibiotic resistance genes: ampicillin resistance and tetracycline resistance. Its useful restriction sites sit in different places:
The question. A gene is inserted at the BamHI site. What phenotype do the recombinants show, and how do you find them?
The reasoning.
Finding them. Plate on ampicillin first — that selects everyone carrying a plasmid and removes the rest. Then test those colonies on tetracycline, by replica plating. Colonies that grow on ampicillin but fail on tetracycline are the recombinants.
The trap, and it catches people every year: the colony you want is the one that does not grow on the second plate. So you cannot simply pick from the tetracycline plate — you have to compare the two plates and go back to the original ampicillin plate to recover the colony, because the replica is a copy you have just killed.
Now invert it. If the insert had gone into the PstI site instead, it would interrupt ampicillin resistance, so recombinants would be ampicillin sensitive and tetracycline resistant, and you would select on tetracycline first. Being able to flip the answer like that is what separates understanding from memorising the diagram.
Blue-white screening replaced double-antibiotic replica plating for routine work because it shows the answer on a single plate.
How it works. The vector carries a fragment of the lacZ gene, and the host supplies the rest; together they reconstitute working β-galactosidase. The plate contains a colourless substrate that the enzyme cleaves into a blue product, plus an inducer to switch the system on. The vector's multiple cloning site sits inside that lacZ fragment.
Now the part the notes leave out. White does not prove you have the right insert. A white colony only tells you the lacZ fragment stopped working. That can happen because:
And a blue colony is not always empty either: a small insert that happens to stay in frame can leave enough function for colour to develop.
There is also a practical trap — small pale colonies crowded around a big one are often satellites that survived because the large colony destroyed the antibiotic nearby. They may carry no plasmid at all. Pick well-separated colonies.
So blue-white is a filter, not a verdict. It tells you which colonies are worth testing further.
This table is the answer to a large share of the marks in this topic, and no set of notes seems to state it plainly.
| Method | What it proves | What it does NOT prove |
|---|---|---|
| Antibiotic selection | The cell contains a plasmid with that marker | Nothing at all about an insert |
| Blue-white screening | The lacZ fragment has been disrupted | That the insert is the right one, or intact |
| Colony PCR | A fragment of about the expected size is present | That the sequence is correct |
| Diagnostic restriction digest | Insert size, and orientation if sites are asymmetric | That there are no point mutations |
| Sequencing | The exact sequence, start to finish | — this is the definitive check |
Read down the table and the logic is plain: each method is stronger evidence than the one above it, and more work. That is why they are used in that order — the cheap test narrows the field, and the expensive test confirms the survivor. An answer that explains that progression scores far better than one that lists the five techniques.
Orientation is worth a sentence of its own. If an insert is ligated with the same enzyme at both ends, it can go in either way round. For a cloning vector that may not matter. For an expression vector it matters completely, because a backwards insert will not be transcribed into the protein you want. Cutting with two different enzymes gives directional cloning, forcing the insert in one way round, and saying so earns a mark whenever expression is mentioned.
A standard exam question gives you a failed result and asks for reasons. Work from the plate backwards.
No colonies at all. Either nothing was taken up or everything died. Check that the host cells were competent, that the heat shock or electroporation step was done correctly, that the cells were allowed a recovery period before being plated on antibiotic, and that the antibiotic concentration was not too high. Also check the obvious: that the plasmid carries resistance to the antibiotic you used.
Plenty of colonies, but all non-recombinant. The vector re-closed on itself. This is why cut vector is usually treated to remove its terminal phosphates, so the two ends cannot be joined without an insert bridging them. Too little insert relative to vector does the same thing.
Colonies on the no-insert control plate. The control is there precisely to reveal this. A heavy lawn on it means your vector preparation was incompletely digested, so uncut circular vector is transforming at high efficiency.
Colonies that look right but the insert is wrong. Partial digestion, star activity from over-digestion, or contaminating DNA. This is exactly what the diagnostic digest and sequencing stages are for.
Always run the controls. Vector with no insert, and cells with no DNA. Without them a failed experiment cannot be diagnosed, and an answer that omits them is incomplete however good the rest is.
See biotechnology help for restriction mapping and ligation calculations, molecular biology help for PCR, primer design and gel electrophoresis, pharmaceutical biotechnology help for transformation efficiency and fermentation, genetics help and microbiology help.
If a cloning question or a failed practical write-up is not coming out, send it over with your own working and you will get an answer from someone who teaches the subject.
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It is the set of methods used to join DNA from different sources and multiply the result inside a host cell. A gene of interest is cut out, joined to a vector, introduced into a host such as a bacterium, and the cells carrying the construct are then selected and screened.
Isolate the DNA of interest and the vector, cut both with restriction enzymes, join them with DNA ligase, transfer the construct into a host cell, and then select and screen for cells carrying what you intended. The marks lie in justifying the choice made at each step.
Selection removes the cells you do not want, usually by growing them on an antibiotic that kills anything without the plasmid. Screening lets you distinguish between the survivors. An antibiotic plate selects; blue-white colour and colony PCR screen. Confusing the two is heavily penalised.
It must replicate independently inside the host, carry at least one selectable marker such as antibiotic resistance, contain unique restriction sites where an insert can be placed without disrupting replication, and be small enough to be handled easily and taken up efficiently by cells.
A cloning vector exists to maintain and copy the DNA sequence itself. An expression vector also carries a promoter the host recognises, a ribosome binding site and usually an induction system, so the insert is actually transcribed and translated into protein. Every expression vector is also a cloning vector.
It is the disruption of a gene caused by inserting foreign DNA inside it. If the insert lands within an antibiotic resistance gene on the vector, recombinant cells lose that resistance while keeping the vector's other marker, which is exactly what allows them to be identified.
Insert at a site inside the tetracycline resistance gene and recombinants become ampicillin resistant but tetracycline sensitive. Select everything on ampicillin first, then replica plate onto tetracycline. The colonies that grow on the first plate but fail on the second are the recombinants.
Because the colonies you want are identified by failing to grow on the second plate. Replica plating transfers them onto a medium that kills them, so the surviving copy exists only on the original. You locate the position on the replica and recover the colony from the first plate.
The vector carries part of the lacZ gene and the host supplies the rest, together making an enzyme that turns a colourless substrate blue. The cloning site sits inside that fragment, so an insert disrupts it. Colonies without an insert go blue; those with one stay white.
No. White only shows the lacZ fragment stopped working. That can happen with the wrong fragment, with several fragments, or with a few bases that shift the reading frame. Small pale satellite colonies may carry no plasmid at all. White colonies must still be confirmed.
The cut vector is usually treated to remove the phosphate groups from its ends, so its two ends cannot be joined to each other. An insert supplying its own phosphates can still bridge them. Using an adequate ratio of insert to vector also reduces self-ligation.
It is cutting the vector and insert with two different restriction enzymes so each end is distinct, which forces the insert in one orientation only. It matters whenever the insert must be transcribed, because an insert ligated backwards will not produce the intended protein.
It screens many colonies quickly for an insert of roughly the expected size, using cells straight from the plate as template. It is fast and cheap, but it confirms only that a fragment of about the right length is present, not that the sequence is correct.
Because a failed result cannot otherwise be diagnosed. A vector-only control reveals incomplete digestion or self-ligation, and a cells-only control reveals contamination or an antibiotic problem. Omitting them leaves you unable to explain what went wrong, and examiners mark that omission.
Yes. A one-to-one tutor can work through vector maps and insertional inactivation with you, check how you interpret screening results, and go over practical write-ups and past-paper questions. You complete your own assessed work in line with your institution's academic rules.
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